Overview

AdvancedFor experienced high school and college classes; requires some technical skill.

Introduce students to the growing field of epigenetics

Gain experience working with the model plant Arabidopsis thaliana

Investigate the role of DNA methylation in gene regulation

Use a protocol designed especially for the classroom

This experiment relates an epigenetic genotype of the FWA gene to a late-flowering phenotype in the model plant Arabidopsis thaliana. The FWA gene encodes a homeodomain containing a transcription factor that controls flowering. Normal flowering requires that both copies of the gene are silenced in the tissues of wild-type plants. FWA is silenced by DNA methylation of repeated sequences in the promoter and 5′ region of the gene. Although fwa-1 mutants have an identical gene sequence, demethylation of this region allows transcription of the FWA gene and causes late flowering. In this laboratory, wild-type Ler and mutant fwa-1 plants are grown, and genomic DNA is extracted from normal and late-flowering plants. The purified DNA is then incubated with the restriction enzyme McrBC. Unlike classical restriction enzymes, McrBC is modification-dependent, recognizing and cutting regions with methylated GC and/or AC dinucleotides. Thus, the methylated, wild-type allele of the FWA gene is cut by McrBC, but the demethylated mutant allele is not. The McrBC-digested DNA and uncut controls are then amplified by polymerase chain reaction (PCR), and the products are analyzed by agarose gel electrophoresis. Primers spanning the FWA promoter and 5′ repeat region amplify a PCR product from the intact DNA from fwa-1 mutant plants but fail to amplify DNA from the digested wild-type plant. Each kit contains materials for 25 DNA extractions and PCR reactions. Note:Order the kit with the perishable materials included (items #211403P, #211404P, and #211405P) or with a prepaid coupon to request perishables later at your convenience (items #211403, #211404, and #211405). Contact us or return the coupon to request delivery of perishable materials. Kit must be used within 4 weeks of receiving it.